Acylation activity

The acylation activities of soluble and immobilized enzymes were evaluated in terms of μmol of octyl octanoate produced per minutes using n-hexane as the acylation medium while using octanoic acid as acyl donor. Briefly, 1.0 g biocatalyst (free or immobilized lipase) was added to 60 mL of reaction mixture containing 1:1 (v/v) octanoic acid and octanol in Teflon stoppered 100 mL conical flask. The flasks were incubated at 30 °C with shaking speed of 120 rpm. The samples were drawn at regular intervals the product concentration was analyzed by HPLC (Agilent Technologies, 11,000 series) equipped with C-18 column (150 mm × 4.6 mm) an autosampler and Diode Array detector (DAD). The mobile phase consisting of 60:40 mixture of methanol: acetonitrile was passed at a flow rate of 1.0 mL min⁻¹. The octyl octanoate eluted was quantified at λ_max 210 nm following calibration curve of standard solution (2–200 ppm; R² 0.9967). One acylation unit of the enzyme was defined as the amount of enzyme that catalyzed the synthesis of one micromole of octyl octanoate per minute (μmol L⁻¹ min⁻¹) under given reaction conditions (Boyall et al., 2002).